19
The Mobile Phase in HILIC
Organic Content
In HILIC the mobile phase is highly organic (generally 60-70%; at least 3% water is required). It has been demonstrated
that besides the selection of a suitable column chemistry, the organic modifier/aqueous ratio is a major factor controlling
the separation selectivity. An increase in the percentage of organic solvent leads to an increase in retention; this
phenomenon is illustrated in the figures below.
*Although Hypercarb is not a polar stationary phase, it was included in this study because of its dual behavior (RP and HILIC), depending on the
percentage of organic in the mobile phase, which allows its use for the retention of polars in RP and HILIC.
For most columns the test compound has little retention (mean k of 0.2) when the acetonitrile content is less than
60%. Acclaim HILIC-10, Acclaim Mixed Mode HILIC-1 and Hypercarb, as seen in the plot on the right display both
typical reversed phase and HILIC mode retention characteristics, depending on the percentage of organic solvent in the
mobile phase. When acetonitrile concentrations are between 60−90%, uracil retention increases with the percentage of
acetonitrile (HILIC mode of retention); between 5−30% acetonitrile, uracil retention decreases as the concentration of
acetonitrile becomes greater (a reversed-phase retention behavior). Interestingly, Acclaim Mixed Mode HILIC-1 does not
retain uracil when the percentage of acetonitrile is between 30−80.
k uracil
% Acetonitrile (v/v)
0.000
0.200
0.400
0.600
0.800
1.000
1.200
1.400
1.600
10
20
30
40
50
60
70
80
90
0
100
Syncronis HILIC
Hypersil GOLD HILIC
Hypersil GOLD Silica
Accucore HILIC
Syncronis Silica
Amide HILIC
k uracil
% Acetonitrile (v/v)
0.100
0.300
0.500
0.700
0.900
10
20
30
40
50
60
70
80
90
0
100
Hypercarb*
Acclaim HILIC-1
Acclaim Mixed Mode HILIC-1
